The development of small organic drugs that mimic protein-protein interactions is an important area for research in biotechnology applications. The use of whole antibodies is not ideal for therapeutic applications because they contain six or more complementary determining region (CDR) loops which can allow for multiple antigen binding sites.2 Although there are only six possible sites, the antibodies developed will have a lower specificity for a particular binding site. This has led to the development of a more suitable, single domain antibody (nanobody), that contains the variable heavy chain domain and only three CDR segments, making them more site specific and therefore more effective.
The production of single domain antibodies or nanobodies is a lengthy procedure that involves multiple steps. First, camelids are immunized with immunogen in both Freund’s complete and incomplete adjuvant.14 The animals then receive 5-6 weekly boosters which helps to generate the high affinity antigen specific heavy chain antibodies.14 This initial immunization is highly efficient and cost effective because the animal is able to be simultaneously immunized with multiple different antigens, allowing for each antigen to develop its own immune response.
Blood samples from the camel are then collected and the peripheral blood lymphocytes are isolated to prepare the cDNA . From previous experiments, it was found that only a small sample (20 -30 mL) from the camelid contained a sufficient amount of β-cells which was required to produce the antigen specific VHH.14
Since the VHH belongs to one single gene family, the VHH repertoire from the immunized animal only needs to be amplified by one set of primers and can be cloned immediately.8 An alternative method involves a secondary polymerase chain reaction with nested primers at the beginning and end of the VHH to produce more material and to restrict certain enzyme sites for cloning purposes.14,15
Once the cloning of the amplified VHH is complete, a VHH library containing the matured antigen binding sites is obtained. A small library of only 106 - 107 VHH individual clones is only required, compared to that of single chain variable fragment antibodies where a larger library is required.14 This is due to the fact that the smaller library allows for the isolation of single domain proteins with nanomolar affinity for their specific antigen. If a small library is not obtained, the immunized camelid will have a higher concentration of binders vs. non-binders, making the process less efficient in the production of antigen binders.14
The process by which VHH libraries are screened for the presence of antigen specific binders is either by panning or by direct colony screening.8,14 The process of panning is preferred over direct colony screening because the panning will also select for those binders with the highest affinity and those that express better in bacteria. From previous experiments it has been found that ~1% of the β-cells contain antigen specific VHH, therefore out of a population of 1000 clones the VHH library is expected to yield ~10 antigen binders.14
Figure 7: Overview for the production process of nanobodies using camelids. (8,14)
The whole procedure, from the first immunization to the selection and identification of VHH nanobodies takes less than three months time.14Despite the success of this process in producing high affinity antigen binders in a fairly short amount of time, the method is sometimes hindered by the initial amount of target antigen required for the initial immunization.16
Production
The development of small organic drugs that mimic protein-protein interactions is an important area for research in biotechnology applications. The use of whole antibodies is not ideal for therapeutic applications because they contain six or more complementary determining region (CDR) loops which can allow for multiple antigen binding sites.2 Although there are only six possible sites, the antibodies developed will have a lower specificity for a particular binding site. This has led to the development of a more suitable, single domain antibody (nanobody), that contains the variable heavy chain domain and only three CDR segments, making them more site specific and therefore more effective.
The production of single domain antibodies or nanobodies is a lengthy procedure that involves multiple steps. First, camelids are immunized with immunogen in both Freund’s complete and incomplete adjuvant.14 The animals then receive 5-6 weekly boosters which helps to generate the high affinity antigen specific heavy chain antibodies.14 This initial immunization is highly efficient and cost effective because the animal is able to be simultaneously immunized with multiple different antigens, allowing for each antigen to develop its own immune response.
Blood samples from the camel are then collected and the peripheral blood lymphocytes are isolated to prepare the cDNA . From previous experiments, it was found that only a small sample (20 -30 mL) from the camelid contained a sufficient amount of β-cells which was required to produce the antigen specific VHH.14
Since the VHH belongs to one single gene family, the VHH repertoire from the immunized animal only needs to be amplified by one set of primers and can be cloned immediately.8 An alternative method involves a secondary polymerase chain reaction with nested primers at the beginning and end of the VHH to produce more material and to restrict certain enzyme sites for cloning purposes.14,15
Once the cloning of the amplified VHH is complete, a VHH library containing the matured antigen binding sites is obtained. A small library of only 106 - 107 VHH individual clones is only required, compared to that of single chain variable fragment antibodies where a larger library is required.14 This is due to the fact that the smaller library allows for the isolation of single domain proteins with nanomolar affinity for their specific antigen. If a small library is not obtained, the immunized camelid will have a higher concentration of binders vs. non-binders, making the process less efficient in the production of antigen binders.14
The process by which VHH libraries are screened for the presence of antigen specific binders is either by panning or by direct colony screening.8,14 The process of panning is preferred over direct colony screening because the panning will also select for those binders with the highest affinity and those that express better in bacteria. From previous experiments it has been found that ~1% of the β-cells contain antigen specific VHH, therefore out of a population of 1000 clones the VHH library is expected to yield ~10 antigen binders.14
The whole procedure, from the first immunization to the selection and identification of VHH nanobodies takes less than three months time.14 Despite the success of this process in producing high affinity antigen binders in a fairly short amount of time, the method is sometimes hindered by the initial amount of target antigen required for the initial immunization.16
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